Roslyn V. Gibbs

Specific recognition of immune cytokines by sulphated polysaccharides from marine algae

Several immune cytokines, including interleukin-2 (IL-2), interleukin-7 (IL-7) and gamma interferon (INF-γ), have been shown to interact with the sulphated polysaccharides heparin and heparan sulphate, so that the co-administration of cytokines with such sulphated sugars may improve current cytokine-based therapies. In this study, sulphated polysaccharides were extracted from the green alga Codium fragile in cold water and purified by size exclusion chromatography. Gas chromatography – mass spectroscopy indicated that they were galactans and/or arabinogalactans, and preliminary nuclear magnetic resonance spectroscopy analysis suggested that they consisted of repeating units of several monosaccharide residues, probably galactopyranose and arabinopyranose, which were sulphated. The binding of Codium fragile polysacchartde and commercial preparations of fucoidan, carrageenans and heparin to IL-2, IL-7 and INF-γ was then assessed using an ELISA-based inhibition test. For all cytokines tested, inhibition by fucoidan was found to be strongest (80–90% inhibition), followed by heparin (65–80% inhibition), the Codium polysaccharide (40-59% inhibition) and finally the carrageenans (20–60% inhibition). The differential binding profiles obtained for the cytokines tested suggest the interactions between cytokines and sulphated polysaccharides are specific and could therefore be exploited in a therapeutic capacity.

A role for chondroitin sulphate B in the activity of interleukin 12 in stimulating γ-interferon secretion

We show, using a murine NK cell line which responds quantitatively to rmIL-12, that treatment with ChABCase, but not other GAGases, results in substantial reductions in the secretion of gamma-IFN. Likewise, treatment of the cells with a beta-D-xyloside inhibitor of proteoglycan biosynthesis inhibits this cytokine response. In both treatments, the addition of soluble, exogenous GAGs does not relieve the inhibition of gamma-IFN secretion. We also demonstrate by ELISA that rmIL-12 binds to CS B. Overall, our studies on this in vitro cellular model of the initiation of Th1 immune responses indicate a major role for cell-surface, iduronate-rich, CS proteoglycan in the biological activity of IL-12.

CYTOKINES AND GLYCOSAMINOGLYCANS (GAGs)

Glycosaminoglycans (GAGs), formerly known as mucopolysaccharides, are anionic polysaccharide molecules that are widely distributed among animal tissues and produced by most cell types. Several key immune cytokines have been shown to bind strongly and selectively to these molecules resulting in modulation of their bioactivity and/or tissue distribution. Thus a greater understanding of these interactions is required to provide insight into the role of GAGs as potential regulators of immune responses.

Micro-heterogeneity and molecular assembly of the haemagglutinins from the red algae Bryothamnion seaforthii and B. triquetrum from the Caribbean Sea

Haemagglutinins have been isolated from two red marine algae Bryothamnion seaforthii and B. triquetrum, which were harvested from the Caribbean Sea, Falcon State, Venezuela. The mass spectra of the haemagglutinins isolated from B. seaforthii showed five monomer peaks (9057.4–9088.8 Da) and those from B. triquetrum showed three peaks (8981.5–8990.2 Da). Isoelectric focusing, under denaturing conditions, gave rise to multiple bands for the haemagglutinins of both species but only one band was observed under native conditions. Gel permeation experiments suggest that the monomer haemagglutinins can readily associate to form dimers, trimers and tetramers. The haemagglutinins agglutinate papainized human A, B and O erythrocytes; the activity is inhibited by glycoconjugates, but not by simple sugars, and is maintained after heating to temperatures up to 90°C. The amino acid sequence of the haemagglutinin from B. seaforthii has been determined together with a partial sequence for that from B. triquetrum. The isolation, and some characterization, of haemagglutinins from B. seaforthii and B. triquetrum, collected from the Ceara coast of Brazil, have previously been reported. Some differences and similarities of these macromolecules obtained from the two different locations are discussed.

Lectins in drug delivery—the binding of some Diocleae lectins to the mucosal surfaces of the eye and mouth

The use of lectins as a means of ‘anchoring’ drug delivery systems to the mucosal surfaces such as those within the oralcavity or precorneal region, in order to enhance localised and systemic drug therapy has been investigated in previous studies (Nicholls et a1 1996, Nantwi et a1 1997). In this work lectins that have been isolated, purified and characterised at the Universidade Federal do Caera, Brazil, were examined for their ability to bind to the buccal and sublingual mucosa, cornea and conjunctiva of the rat, with regard to their potential for inclusion into new drug delivery systems. The lectins from Canavalia ensiformis, Canavalia brasilensis, Canavalia bonariensis, Cratylia floribunda, Dioclea grandiflora, Dioclea guianensis, Dioclea violacea, Dioclea virgata and Dioclea rostrata were investigated in this study. The Canavalia ensiformis lectin supplied by Sigma was used as a control. These lectins were biotinylated using biotin N-hydroxysuccinimide ester and a technique developed at the University of Portsmouth. The molecular weights of the lectins were determined before and after biotinylation, and from this biotin : lectin (or lectin subunit) ratios were estimated by SDSPAGE to be between 2.4:l (Dioclea rostrata) and 10: 1 (Canavalia brasilensis). Uppereyelids, eyeballs, buccal tissue and tongues were obtained from recently sacrificed male wistar rats. These yere exposed to solutions containing 5pg mLbiotinylated lectins for 15 min, washed and the lectin binding identified using a streptavidin peroxidase/diaminobenzidine technique as described by Nicholls et a1 (1996). 5pm sections were cut and the surface cover and stain intensity of the diaminobenzidine precipitate, indicting the presence of bound lectin, was subjectively assessed for surface cover (0 to 5, from no cover to complete coverage) and stain intensity (to +++++, from no visible precipitate to a dark brown precipitate) using light microscopy at x 100 magnification. The experiment was completed using tissues from three different animals for each lectin.

The localisation of the heparin binding sites of human and murine interleukin-12 within the carboxyterminal domain of the P40 subunit

Graphical abstract Figure. No Caption available. HighlightsWe demonstrate differences in the specificity of heparin binding between human and murine IL‐12s.Heparin predominantly protects the p40 subunit against proteolysis by LysC.A truncated IL‐12 polypeptide lacking the carboxyterminal D3 domain fails to bind heparin.The C′D′ loop of the D3 domain contains a large cluster of surface accessible basic residues.This loop is the region of greatest sequence variation between murine and human p40s. &NA; We have previously shown that the heterodimeric cytokine interleukin‐12, and the homodimer of its larger subunit p40, both bind to heparin and heparan sulfate with relatively high affinity. In the present study we characterised these interactions using a series of chemically modified heparins as competitive inhibitors. Human interleukin‐12 and p40 homodimer show indistinguishable binding profiles with a panel of heparin derivatives, but that of murine interleukin‐12 is distinct. Heparin markedly protects the human and murine p40 subunits, but not the p35 subunits, from cleavage by the bacterial endoprotease LysC, further implicating the larger subunit as the location of the heparin binding site. Moreover the essential role of the carboxyterminal D3 domain in heparin binding is established by the failure of a truncated construct of the p40 subunit lacking this domain to bind. Predictive docking calculations indicate that a cluster of basic residues at the tip of the exposed C′D′ loop within D3 is important in heparin binding. However since the human and murine C′D′ loops differ considerably in length, the mode and three dimensional orientation of heparin binding are likely to differ substantially between the human and murine p40s. Thus overall the binding of IL‐12 via its p40 subunit to heparin‐related polysaccharides of the extracellular matrix appears to be functionally important since it has been conserved across mammalian species despite this structural divergence.